From:
Rev.Physiol. Biochem.Pharmacol. (2003) 148:1–80
F.Wehner·H.Olsen·H.Tinel·E.Kinne-Saffran·R.K.H.Kinne
Cellvolumeregulation:osmolytes,osmolytetransport, andsignal
AbstractInrecentyears,ithasbecomeevidentthatthevolumeofagivencellisanimportantfactornotonlyindefiningitsintracellularosmolalityanditsshape,butalsoin definingothercellularfunctions,suchastransepithelialtransport,cellmigration,cell growth,celldeath,andtheregulationofintracellularmetabolism.Inaddition,besidesinorganicosmolytes,theexistenceoforganicosmolytesincellshasbeendiscovered.Osmolytetransportsystems—channelsandcarriersalike—havebeenidentifiedandcharacterizedatamolecularlevelandalso,toacertainextent,theintracellularsignalsregulating osmolytemovementsacrosstheplasmamembrane.Thecurrentreviewreflectsthesedevelopmentsandfocusesonthecontributionsofinorganicandorganicosmolytesandtheir transportsystemsinregulatoryvolumeincrease(RVI)andregulatoryvolumedecrease(RVD)inavarietyofcells.Furthermore,thecurrentknowledgeonsignaltransductionin volumeregulationiscompiled,revealinganastonishingdiversityintransportsystems,as wellasofregulatorysignals.Theinformationavailableindicatestheexistenceofintricate spatialandtemporalnetworksthatcontrolcellvolumeandthatwearejustbeginningto beabletoinvestigateandtounderstand.
Generalintroduction
Foranumberofyears,cellvolumeregulationinmammaliancellshasbeenconsideredof minorphysiologicalimportancesinceoverallosmolalityoftheplasmaisoneofthevery tightlyregulatedparametersofthebody.Inthemeantime,however,ithasbeenrealizedthatquiteavarietyoftissuessuchasrenalmedullarycellsandchondrocytesareexposed toanisotonicextracellularmediaandthusrequirevolumeregulatorymechanisms.Anisotonicconditionscanalsoariseinpathologicalconditionssuchashypo-orhypernatraemia whenhomeostaticfunctionsofthebodyareinsufficient.Similarly,cellsencasedinarigidsurrounding,suchasbrain cells, depend on cell volume regulation fortheir proper function.Furthermore, even atnormalextracellularosmolalitiescellscangeneratetransmembraneosmoticdifferencesdue touptakeof(organic)osmolytesorduringtransepithelialtransportofsolutes.
Cellvolume changes alsooccurglobally during maturationof erythrocytes,cellgrowth, differentiation,hypertrophy,andapoptosis.Locally,cellmigrationandshapechangesrequirevolumeadaptations.Finally,cellvolumehasbeenidentifiedasa mechanismthatregulatescellmetabolism.Thiseffectisparticularlyevidentintheliver,wherecellswellingincreasesproteinandglycogensynthesisandcell shrinkageincreasesproteinandglycogenbreakdown. Thus,cellvolumeanditsimportanceduringthewholelifecycleofacellhasbecome moreandmoreevidentandthisareaofphysiologyandpathophysiologyhasbeenattractinganincreasingnumberofinvestigators.
Duringrecentyears,italsobecamemoreandmoreacceptedthatnotonlyinorganicosmolytessuchasK+andCl-areemployedtorestoreosmoticequilibriumacrossthecell membranesbutthatalsoorganicosmolytesplayasignificantroleincellvolumehomeostasis.
Inmostreviews,inorganicandorganicosmolytesandtheirrespectivevolumeregulato-rytransportersare dealtwithseparately.Thecurrentreviewcombinesthetwoanddiscuss-esthequestionofaputativeidentityofsomesystems.Thiscontributionalsotakesinto accounttheprogressmadeinidentifyingthemolecularentitiesofthetransportersbymo-lecularbiologytechniques.Furthermore,thecurrentstateofknowledgewithregardtothe regulationandcoordinationofthevariousosmolytetransportersisreviewedinorderto shedsomelightontheintriguingsignaltransductionnetworksusedincellvolumeregula-tion.
Theosmometricbehaviorofcells
Anychangesincellvolumeofanimalcellsunderanisotonicconditionsare,inprinciple, basedonadistinctpermeabilityoftheplasmamembranetowater.Inmanyinstances,this permeabilityisincreasedbyspecializedmembraneproteins,whichmediatethefacilitated diffusionofwater,so-calledwaterchannelsoraquaporins.Givenasizeablewaterpermeability atitsoutermembrane,acellthenexhibitsan“osmometric”behaviorasitsfirstpassive responsetoanisotonicity,i.e.,amovementofwaterwilloccur,whichchangescellvolume untilthedifferenceinosmoticpressureisequalizedandanewchemicalequilibriumof wateracrosstheplasmamembraneisachieved.Fromaphysicochemicalpointofview, however,acellisnotaperfectosmometer,whichmeansthatitsvolumewillnotchange tothesameextentasispredictedbythelawofBoyle/van’tHoff.Thismeansthatappar-entlysome20%–40%oftotalcellvolumeusuallycomprisesaphasethatisnot“cytosolic,”whichmeansthatitisnotosmoticallyactive(LuckeandMcCutcheon1932).Inpractice,theosmoticallyactive/inactivespaceofalivingcellcanbeeasilydeterminedgraphicallybyplottingitsrelativevolumeversusthereciprocalofextracellularosmolarity, whichisequivalenttoaBoyle/van’tHoff-Plot(seeFig.1).
TheinterceptoftheregressionlinewiththeY-axisthengivestheosmoticallyinactive space;itsdifferencefromthe100%markisequivalenttotheosmoticallyactivepart.This typeofanalysis,whichdealswiththepassiveosmoticbehaviorofacell,however,isonly validiftheactivecompensatoryprocessesbywhichcellsreadjusttheirvolumearenotactivatedand/orareinhibitedefficiently.Inrathepatocytes,forinstance,thiswasachieved byblockingallNa+importmechanismsmediatingregulatoryvolumeincrease(RVI)(namelyNa+conductance,Na+/H+antiport,andNa+-K+-2Cl-symport)by10-3Mol/lamilorideand10-4Mol/lfurosemideandbythendeterminingtheactualchangesofcellvolumeafter10minofcellshrinkage,underfourdifferenthypertonicconditions.Fromthese measurements,anosmoticallyinactivespacecouldbedeterminedthatamountstosome31%oftotalcellvolume.
Inorganicandorganicosmolytes
Foranumberofyears,researchonvolumeregulationinmammaliancellshasfocusedon themovementofthemainintracellularinorganicosmolytessuchaspotassiumandchloride.Compensatorychangesintheseosmolytesformindeedthebackboneforvolumeregulationwhenonlysmallperturbationsoftheintra-orextracellularosmolarityoccur.
Inrecentyearsithasbecome,however,moreandmoreevidentthat—similartoother phylainnature—inmammaliancellsalsoorganicosmolytesareemployedinvolumeregulation.
Organicosmolytescomprisepolyolssuchassorbitolandmyo-inositol,methylamines suchasglycerophosphorylcholineandbetaine,andaminoacidssuchastaurine,glutamic acid,andß-alanineamongstothers.
Thereareseveralhypothesesastowhyinsomecellsorganicsolutesareusedinadditiontoinorganicsolutes.Onehypothesisisthe“compatibleosmolytehypothesis”whichisbasedontheobservationthathighconcentrationsofinorganicsaltssuchasNaClor KClperturbthefunctionofenzymesorotherproteins,whereasorganicsolutesdonot.Anotherhypothesisreflectsthe“counteractingosmolyteprinciple,”whichreferstothefindingthatmethylaminesattenuatethedestabilizingeffectofhighconcentrationsofureaonproteinfunction.Athirdaspectis thatseveralorganicosmolytesareelectroneutralandcanreplaceinorganicosmolytes which,whenreleasedacrossthecellmembrane,maychangethemembranepotentialand therebyneuronalexcitabilityinthebrainordrivingforcesforelectrogenicsodium-cotransportsystems.Again,thebrainisparticularlyinterestingbecauseof theexistenceofreuptakesystemsforexcitatoryaminoacidsthataretransportedbysodium-cotransportsystems.
Oneofthemajorplacesfororganicosmolyteaccumulationistherenalmedulla,where accumulationoccursbecauseofabroadrangeofextracellularosmolalitiesexceedingnormalosmolalityinparticularinthedirection ofhyperosmolality.
Intracellularaccumulationoforganicosmolytesinvolvesmainlytwoprocesses.The firstrepresentsuptakeacrossthecellmembranebyspecifictransportsystems,thesecond intracellulargenerationoftheosmolytebymetabolicreactions.Examplesoftheformer aresodium-cotransportsystemsformyo-inositol,taurine,andbetainewhichusethesodi-umgradientacrosstheplasmamembraneasdrivingforce.Thegeneralandspecificpropertiesofthesecotransportsystemsaresummarizedinthesectionentitled“Organicos-molytesinRVI.”
Regulatoryvolumeincrease
RoleofinorganicosmolytesinRVI
InordertoachieveRVI,theintracellularosmolytecontenthastobeaugmentedrapidly. Tothisend,transportsystemsforinorganicosmolytesareactivatedasthefirstcellularresponse.Themainosmolytetakenupbythecellsissodiumsincefavorabledrivingforces forthiscationexistacrosstheplasmamembrane.Sodiumissubsequentlyexchanged againstpotassiumbytheactionoftheNa+,K+-ATPase,torestoretheoriginalsodiumgradientsanditselectrochemicalpotential.
Therearealsomechanismssetinplacethatreducelossofintracellularpotassiumand sometimesalsochloride,dependingonitselectro-chemicalequilibrium.
Na+/H+antiport
Na+/H+antiporters(NHEs)catalyzethesecondaryactiveandelectroneutralexchangeof H+againstNa+.Withoneexception,theyaremostimportantfortheregulationofcellpH, butsomeNHEsareeffectivemediatorsofRVI.SixNHEisoformshavebeenclonedto date(NHE1–NHE6)whichexhibitacommonmolecularorganization.
Withrespecttotheirroleincellvolumeregulation,itwasfoundthatNHE1,NHE2, andNHE4areactivatedunderhypertonicconditions,whereasNHE3isinhibited.NHE1isclearlythe Na+/H+antiportmostcommonlyemployedinRVI,whichisnotsurprisinginregardtothe ubiquitousexpressionoftheprotein.Asamatteroffact,theshrinkage-inducedactivation ofNa+/H+antiportthathadbeenreportedinavarietyofpreparationspriortothemoleculardefinitionofNHEisoformscannowinmanyinstancesbeattributedtoNHE1.
Na+-K+-2Cl-symport
NKCC1andNKCC2aremediatorsofelectroneutralNa+, K+, Cl-cotransportacrosscell membranesatastoichiometryof1:1:2.Onthemolecularlevel,theysharemanysimilarities withothermembersofCl--dependentcationtransporters,namelytheNa+-Cl-symporter
(NCC)andthefourisoformsofK+-Cl-symporters(KCC1toKCC4)clonedsofar.These similaritiesincludemolecularweightsintherangeof110kDto130kD(deglycosylated),
12predictedtransmembraneregions,andlargehydrophilicintracellularN-andC-terminal domains.Themostconservedregionsofthesetransportersarethetransmembranedo-mains,aswellastheputativeintracellularloopsconnectingthem,particularlytheonebe-tweenTM2andTM3.NKCCsareblockedbythe“loop”diureticsbumetanideandfurose-mideatmicromolarconcentrations;KCCsexhibitaloweraffinitytothesecompounds, whereasapossibleinhibitionofNCCremainsambiguous.NKCC1exhibits abroadtissuedistributionandisfoundinmanysecretoryepithelia(whereitresidesinthe basolateralmembrane),aswellasinavarietyofnonepithelialcells.Incontrast,NKCC2 isonlypresent(apically)inthethickascendinglimbofHenle’sloopandinthemacula densaofthekidney(HaasandForbush,III2000;Russell2000).
Inrathepatocytes,thehypertonicactivationofNa+-K+-2Cl-symport(aswellasthatofNa+conductance)wasinhibitedbystaurosporine,aswellasbythePKCspecificblockerbis-indolyl-maleimideI(BIM;Heinzingeretal.2001).Inhumantrachealepithelialcells,thehypertonicactivationofNKCC1appearedtobemediatedbyPKC-d;thisprocessisalsolikely toinvolvethe(extracellularsignal-regulated)kinaseERK(LiedtkeandCole2002).Inaddition to phosphorylation anddephosphorylation, the activity ofNKCC1appearsto be reg-ulatedbythestateoftheactinnetwork,aswellasbyaccessoryproteinsthatremaintobe characterized(HaasandForbush,III2000;Russell2000).
Cationchannels
Comparedtothesymportsystemdiscussedabove,theratesofiontransportbychannels aresome4–5ordersofmagnitudehigher.Accordingly,anymodulation ofchannelactivity inresponsetochangesofcellvolumewillserveasafastandveryefficientregulatory mechanism.
IfoneconsiderstheelectrochemicaldrivingforcesforNa+, K+,andCl-transportacross mostcellmembranes,theactivationofNa+-selectivechannelsandconductiveNa+entry wouldbeahighlyefficientmediatorofRVI.Theresultantdepolarizationofmembrane voltagewouldfavoraparallelconductiveentryofCl-. Na+accumulatinginsidethecell wouldthenbeextrudedviaNa+, K+-ATPasesothatconstantdrivingforcesforNa+-cou-pledcotransportersareensured.Insum,anetintracellulargainofK+andCl-andthusa rapidincreaseofcellvolumewouldbeachievedbythesemechanisms.
Thereisanincreasingnumberofsystemsfromwhichthehypertonicactivationofcationchannelsisreported.Twomainclassesofchannelscanbedistinguishedbasedontheir sensitivitytoamiloride.Inthefollowing,firsttheamiloridesensitivechannelswillbediscussed.
TheactivationofconductiveNa+entryasamechanismofRVIwasoriginallyproposed byHoffmannforEhrlichascites tumorcellsandtheIntestine407cellline,respectively.In1995,ahypertonicstimulation ofcellmembraneNa+conductancewasreportedfromcurrent-clamprecordingsonrathepatocytesinconfluentmonolayerculture(seeFig.2).
Fig. 2 A Cable analysis of specific membrane resistance, reflecting the hypertonic activation of Na+ conductance in rat hepatocytes. Experiments were carried out in the continuous presence of 0.5 mM quinine; extracellular osmolarity was increased from 300 mOsm/l to the osmolarities indicated; means€SEM; n=4–5. B Current-voltage relationships of hypertonicity-induced membrane currents in rat hepatocytes obtained with (two-channel microelectrode) voltage-clamp techniques. The differences between currents obtained at 400 mOsm/l and 300 mOsm/l are depicted for cells injected with control-oligo-DNA ()or anti-rENaC oligo DNA ().
Furthermore,inaquantitativestudy,itcouldbeshownthattherelativecontributionof Na+conductance,Na+/H+antiport,andNa+-K+-2Cl-symporttotheinitialuptakeofNa+ underhypertonicstresswasapproximately4:1:1.ThisclearlyrendersNa+conductancetheprominentmechanismofRVIinthissystem.
Inhumanredbloodcellghosts,hypertonicstressinducedacationconductancethatappearedtobeequallypermeabletoNa+andK+(butimpermeabletoNMDG)andthatwas partiallyinhibitedby10-4Mol/lamiloride(Huberetal.2001).Thesefindingssupportearlierfluxstudiesonlampreyerythrocytesinwhichhypertonicstressledtotheactivationof anamiloride-sensitiveNa+transportthatcouldnotbeattributedtoNa+/H+orNa+/Na+an-tiport.
K+channels
Inmostsystems,thecontinuouschannel-mediatedK+leakageoutofcellsisequivalentto asignificantlossofcellularosmolytes.ThisK+lossiscommonlycompensatedforbythe activationofNa+, K+-ATPase.Nevertheless,itisratherobviousthatanyinhibitionofK+ channelsunderhypertonicconditionswillpersefacilitateRVIand,insomecells,this mechanismappearstobeanimportantmediatorofvolumeregulation.
AninhibitionofK+conductancewasoriginallyreportedforthebasolateralmembrane oftoadandrabbiturinarybladder,aswellasof frogskinwherecellswereshrunkenbyextracel-lularCl-removal(leadingtoalossofCl-becauseofthelowermembranepermeabilityof theCl-substituteusedwhencomparedtoCl-itself)and/orbyincreasingextracellularos-molarity.Interestingly,whole-cellrecordingsonfreshlyisolated hippocampalneuronsrevealedadecreaseofvoltage-gatedK+currentsunderhypertonic conditions.
Anionchannels
InhibitionofanionchannelsduringRVImightdirectlycontributetothegaininosmolytes iftheintracellularanion(Cl-orHCO3-)activityisaboveelectrochemicalequilibrium.In-directly,itrestrictsthemovementofthecounterionforK+fluxacrossthemembrane,thus impedingK+losses.
DecreasesofCl-conductanceunderhypertonicconditionshavebeenreportedonly fromalimitednumberofpreparations.Inculturedhumannasalepithelium,hypertonic stressreducedapicalCl-conductance whereas,inrabbitcollapsed proximalconvolutedtubules,thepartialCl-conductanceofthebasolateralmembranewas inhibited.Insomeinstances,thedecreaseofCl-conductanceunder hypertonicconditionsmayreflectaninhibitionofhypotonicity-inducedCl-channelactivation.Inhumanvasdeferenscellsinprimaryculture,forexample,with290mOsm/lsolutionsintheexperimentalbath,aswellasinthepatchpipette,therewasaslowlydevelopingincreaseofCl-conductanceoncethewhole-cellconfigurationwasachieved;this effectcouldbereversedbyanincreaseofextracellularosmolarity(Winpennyetal.1996). TheincreaseofCl-conductancewasinterpretedtobeduetotheadditionalosmoticactiv-ityofintracellularmacromoleculesandtheresultantswellingofcells.
OrganicosmolytesinRVI
Inmostcellsinvestigated,RVIafterexposuretoahypertonicextracellularmediumoccurs withinminutesandinvolvesthenetuptakeofinorganicosmolytes.Duringthisshort-term regulationofcellvolume,changesinorganicosmolytestransportaregenerallynotinvolved,althoughithasbeenobservedthattheplasmamembranepermeabilityfororganic osmolytessuchassorbitoldecreases. Thus,leakpathwaysfororganicosmolytesaredownregulatedtomaketheintracellularaccumulationoforganicosmolytes—bytransportersormetabolicsynthesis—moreeffective. WhethertheseleakpathwaysareidenticaltotheonesactivatedduringRVDremainstobe investigated.
Ifexposureofcellstohypertonicityisextendedtoperiodsofhoursordays,adaptive changestakeplaceaimedtoreplacetheaugmentedintracellularinorganicelectrolytesby organicosmolytes.Tothisend,intracellularsynthesisisincreased(sorbitolandGPC)or intracellularbreakdownisdecreased. Fortaurine,betaine,andmyo-inositol,thepredominanteffectisontherateofuptake, whichincreasesseveralfoldinamatterofhours(seeFig.3).
Fig. 3 Time course of basolater-al myo-inositol (A) and betaine (B) uptake into MDCK (Madin-Darby canine kidney) cells switched into hypertonic medi-um. On day 0, cells cultured on filters in defined medium with 10% FBS were switched to same medium made hypertonic (500 mOsm/l) by addition of raf-finose. Isotonic cells were main-tained in isotonic defined medi-um with 10% FBS. Uptake was performed at 37 C for 30 min with 10 Mol/l myo-inositol and 10 Mol/l betaine. Results are means€SD to triplicate samples (from Yamauchi et al. 1991)
Thus,strictlyspeaking,organicosmolytesarenotdirectlyinvolvedinRVI;theyserve, however,inthelongruntomaintaincellularelectrolytehomeostasis.Therefore,thecellu-laruptakesystemsfororganicosmolytesandtheirregulationaredescribedinthefollow-ingparagraphs.
Na+-Cl--taurinesymport
Taurine,a-aminoacidwithasulfonicacidinsteadofacarboxylicacidasheadgroup occursinplasmaatconcentrationsof40Mol/l.Ithasseveralmajorfunctions:aspartner intheformationoftaurocholate,asneurotransmitter,andasanorganicosmolyte.Althoughseveraltissues,includingliver andastrocytes,cansynthesizetaurinefromcysteine,thehighintracellularconcentrationcanonlybeachievedbyanactiveuptakeintothecell.Thetransportermediatingthisuptakeisasecondaryactivesodium-chloridetaurinecotransportsysteminwhichuptothreesodiumionsaretranslocatedwith onechlorideionandonetaurinemolecule.Taurinetransportwiththesecharacteristicshas beenfoundinrenalandintestinalbrushborder,inlivercells.
InavarietyofcellsandtissuessuchasMDCKcells,humanand bovinelensepithelialcells,bovineaorticendothelialcells,ratlivermacrophages(Warskulatetal.1997a),H4IIEhepatomacells
(Warskulatetal.1997b),primaryculturesofrathepatocytes,rat astrocytecultures, exposuretohyperosmoticmediaresultsinanincreaseinVmaxoftaurinetransportanda concomitantincreaseintransportermRNA.
HighaffinitytaurinetransportisinhibitedbyphosphorylationviaPKCbydecreasing affinityfortaurineandsodiumandreducingmaximalvelocity.Althoughtheseregulatoryprocessesappearnottobelinkedtotheresponsetohypertonicity,theycomplicate thestudyofthemolecularmechanismsofthelatter.However,itcanbeassumedthatthe mechanismsinvolvedinosmoticadaptationaresimilartotheonesdescribedbelowforthe betainetransportsystem,sincebothsystemsrespondinaverycomparablemanner.
Na+-Cl--betainesymport
Inmedullarykidneycellsandchondrocytes,whichusebetaineinvolumeregulation,intracellularconcentrationsofupto50mMol/larefound.Althoughcellsusuallycontaincholinedehydrogenasethatcatalysesthesynthesisofbetainefromcholine,mostofthebetaineistakenupfromtheextracellularmedium.
Hypertonicactivationofbetaineuptakeshowssomemorepeculiarities.Underisotonic conditionsbetaineuptakeintoMDCKcellsproceedsonlyacrossthebasolateralmem-brane;afterexposuretohypertonicity basal-lateraluptakeisincreased,but inaddition, sig-nificantuptakeacrosstheapicalmembraneisobserved(Yamauchietal.1991).Thisfind-ingmightberelatedtotheexistenceofvariousisoformsofBGT1(seeabove)thatmight differintheircellularlocation.
Na+-Myo-inositolsymport
Physiologicalplasmaconcentrationsofmyoinositolrangeinmammalsfrom4.5mMol/l to6.6mMol/l,whereasintracellularconcentrationsupto133mMol/lcanbefoundinrat glial.Thislargeconcentrationdifferencesuggestsactiveuptakeofmyo-inositolinto thesecells.Similarly,activemyo-inositoluptakeisobservedinratpancreaticislets,bovinelensepithelialcells,hamstersmallintestine,ratmesanglialcells,rathepatocytes, crystallinelense,rabbitperipheralnerve,retinalpigmentepithelialcells,rabbitciliarybody,isolatedratSchwanncells,andendothelialcells.Thisuptakeprocessinvolvesasodium/myo-inositolsymportsystem(SMIT)which assecondaryactivetransportsystememploysthetransmembraneelectrochemicalpotentialdifferenceofsodiumforintracellularaccumulationofmyo-inositol.
Thesodiumtomyo-inositolstoichiometryofthetransporteris2:1. Thestoichiometryandelectrogenicityofthetransporterformsthebasisfortheextensive intracellularaccumulationofmyo-inositol.Kineticandbiochemicalcharacteristicsvary fromtissuetotissueandcelltocell.Thesevariationshaverecentlybeenexplainedbythe existenceofalternatesplicing,whichgeneratesisoformsthatdifferintheirintracellular proteinkinaseAandproteinkinaseCphosphorylationsitesatthecarboxyterminus(Por-cellatietal.1999).
Afterrestoringisotonicity,thetransportratereturnstonormallevelswithin1d.Inboth instancesonlytheVmaxofthetransportisaffectedandKmremainsconstant,suggestinga changeinthenumberoftransporters.Theincreaseduptakeinresponsetohypertonicityispreceded byanincreasedabundanceofmRNAforthetransporter,whichisthedirectresultofincreasedtranscriptionofthegene.Activationoftranscriptiondependsonanenhancerelementnamedtonicityresponsiveenhancer(TonE).
AminoacidtransportsystemA
AminoacidtransportsystemA(SystemA),whichmediatessodium-dependentuptakeof neutralaminoacidsintomammaliancells,appearstobealsosubjecttoregulationbyex-tracellularosmolality.InMDCKcells,aswellaschondrocytes,anupregulationwithin4–6hofhypertonicexposurewasobserved.Theincreasein SystemAisblockedbyinhibitorsofRNAandproteinsynthesis,suggestingthatanincreaseinthenumberoftransportersispartofthemechanism.Theadap-tationofthissystemappearstobearelativelyearlyresponseofcellsexposedtohyperto-nicity;therefore,itdeservesfurtherinvestigation.
Regulatoryvolumedecrease
InorganicosmolytesinRVD
Aftercellswelling,transportsystemsareimmediatelyactivatedthatmediatethereleaseof themajorintracellularinorganicosmolytespotassiumandchloride.Theirtransmembrane movementoccurseitherviaseparatepathwaysordirectlycoupledtoeachother.
K+channels
Due tothe outwardlydirectedK+gradientinmost animal cells,anyincreaseof K+channel activitywillaugmenttheconductiveexitofK+.Inaddition,theincreaseofcellmembrane K+conductancewillhyperpolarizethecellmembraneand(evenifbasalCl-conductance isnotchanging)thishyperpolarizationwillfavorconductiveCl-efflux.Likewise,ifthere isaninitialincreaseofCl-conductanceandifcellCl-isaboveelectrochemicalequilibrium(asitisinmostsystems)thiswillaugmentCl-exitanddepolarizethecellmembrane. MembranedepolarizationinturnwillfacilitateconductiveK+efflux.Themosteffective mechanismofRVDwill,ofcourse,betheparallelactivationofK+andCl-channels.Becauseofthepronouncedvoltage-mediatedcouplingbetweenbothpathwaysconductiveK+ andCl-releasemayresultinaquasielectroneutralmodeofKClexport.
AnincreaseofcellmembraneK+conductanceunderhypotonicconditionsanditssignificanceforRVDhavebeenreportedfromavarietyofpreparations.
BKCa ormaxi-K+channelsexhibitlarge(big)unitaryconductancesintherangeof some100pSto250pSandshowundersymmetricalhighK+solutionsalinearcurrent-to-voltagerelation.Inmostinstances,BKCachannelsare inhibitedbyBa++,quinine,andTEA(tetraethylammonium),aswellasby(thescorpion peptide)charybdotoxin.Theyareselectivelyblockedby(thescorpiontoxin)iberiotoxin. BKCachannelsarefoundinneurons,skeletalmuscle,smoothmuscle,andinepithelial cells,wheretheyresideintheapicalmembrane.BKCachannelsareactivatedbymembranedepolarizationandmicromolarconcentrationsofcellCa2+.ItisthisCa2+sensitivity thatmightfunctionasalinktoaswelling-inducedincreaseinintracellularcalcium.Likewise,thevoltage-dependenceofBKCachannelsmayreflectthecouplingmechanismtoa hypotonicity-inducedactivationofCl- channels.Theirmembranetopologyis relatedtothatofvoltage-gatedK+channels.
TheactivationofBKCachannelsunderhypotonicconditionscouldbedemonstratedin Necturusandrabbitproximaltubulecells,inprincipalcellsofrabbitandratcorticalcollectingtubule,inclonal kidneycellsderivedfromAfricangreenmonkey,inacinarcellsofratlacrimalgland(Parketal.1994),inembryonicchickhepatocytes,andinguineapigjejunalvillusenterocytes.
Theseresultssuggestadirecteffectofmembranestretchonthechannelitselforona closelyassociatedcomponentasamodeofchannelregulation.Alsoofnoteisthedistinct sensitivityofBKCachannelstoextracellularATPasitwasobservedinVerocells. Thus,ATPrelease maywellfunction as an autocrine(orparacrine) mechanismofhypotonicchannelactivation.
IKCachannelsareactivatedbycytosolicCa2+activitiesintheuppernanomolarrange andexhibitanintermediateconductancethat(at0mV)equals20pS–80pS.Theyare voltage-independentbutweaklyinwardlyrectifying(undersymmetricalhighK+conditions).IKCachannelsareweaklyinhibitedbyquininebutefficientlyblocked bycharybdotoxinand(moreselectively)byclotrimazole.
ThehumanIKCa channelis88%and90%identicaltothemouseandratchannel,respectively,and40%to42%identicaltoSKCachannels.
Thereareseveralreportswhichidentifythistypeofchannelasthemediatorofconductivehypotonicity-inducedK+release.InthehumanepithelialcelllineIntestine407,hypotonicswellingledtoasignificantincreaseofcellCa2+,therebyactivatingIKCachannels.HumanTlymphocytesexpressaCa2+-activatedK+conductancethat wasactivatedbyhypotonicconditions(Khannaetal.1999).TheK+conductancewas slightlyinwardlyrectifyingandblockedbycharybdotoxinaswellasclotrimazole.
SKCachannelsundersymmetricalhighK+conditionsexhibitasmallconductanceof some4pS–18pS(at0mV)andareactivatedbynanomolarconcentrationsofCa2+.They areinwardlyrectifyingandvoltage-independent.In electricallyexcitable cells,SKCachannelsmediatethe slowafter-hyperpolarizationfollowingactionpotentials.
Thusfar,thereisonlylimitedevidencefortheinvolvementofSKCainvolumeregulation.
Twomembersofvoltage-gatedK+channelscouldbedefinedasmediatorsofRVD, namelyKv1.3andKv1.5.KvchannelswerefoundtobealsoexpressedinelectricallynonexcitablecellssuchasT-lymphocytes.Thereisincreasingevidencesuggestingthattheyplayaroleincellvolumeregulation(Felipeetal.1993;Lewis andCahalan1995).Itwasfound,forinstance,thatamouseT-lymphocytecell-line(CTLL-2)thatdoesnotexpressvoltage-dependentK+channelsdidnotexhibitanysignifi-cantRVDunderhypotonicconditions.
Cl- channels
Theapparentlymostabundantformofhypotonicity-inducedCl-channelsbelongtoa groupthathasbeennamedVSOR(volume-sensitiveoutwardly-rectifying)Cl- channels,VRAC(for“volume-regulatedanionchannels”,orVSOAC(volume-sensitiveorganicosmolyte-anionchannels).
An exampleforsuchachannelisgiveninFig.4.
Fig. 4 AActivationofanionconductanceinratinnermedullarycollectingduct(IMCD)cellsunderhypo-tonicconditions.Whole-cellpatch-clamprecordingwithsymmetricalCsClsolutions;theosmolarityofthe pipettesolutionwas600mOsm/l.Atthetimeindicated,osmolarityofthesuperfusatewasreducedfrom600mOsm/lto500mOsm/l.Voltagerampsfrom-60to+60mVand2sdurationwereappliedevery15s. BCurrent-voltagerelationshipbefore(linea),at6.5min(lineb),andat11min(linec)ofhypotonicity; currentswerereferredtomembranecapacitance. CTime-dependentinactivationofhypotonicity-induced Cl-conductanceatdepolarizingvoltages.Membranevoltagewassteppedfrom-80mVtopositivevalues in the range of +30 to +120mV with 10 mV incrementafter each pulse. Pulsedurationandinterval between pulseswere10 sand 15s,respectively(Aand BfromBoeseetal.1996b;CfromBoese etal.1996a)
TheirsignificantoutwardrectificationpersistsundersymmetricalCl-conditions.An interestingfeatureof VSORCl-channelsistheirpronounced inactivationwhenmembrane voltageissteppedfromverynegativevaluestoabove+50mV.
ClC-2andClC-3werealsoconsideredasmolecularcandidatesforVSORCl- channels. Thefirstmemberofthisgroupofvoltage-gatedchannels(ClC-0)wasclonedfromthe electricrayTorpedo(Jentschetal.1990).Typically,ClCsexhibittentotwelvetransmem-branehelicesandtheN-andC-terminiarelocatedinthecytosol.MostClCs(exceptsome prokaryoticisoforms)containtwohydrophobicdomainsclosetotheC-terminusthatmay beinvolvedintheircellularsorting(Schmidt-RoseandJentsch1997;Jentschetal.1999). ClCsweresupposedtofunctionasdimers,witheachmonomershowingaseparatepore.
ClC-3exhibitsmanysimilaritiestoVSORCl- channelsincludingoutwardrectification, unitaryconductance,anionselectivity,voltage-dependentinactivation,andinhibitionby extracellularnucleotides,stilbenederivatives,andtamoxifen.Therearesomesignificantdifferencesremaining,however. AmongtheseistheenormousbasalactivityofClC-3whencomparedtoVSORCl-chan-nels,thelatterbeingundetectableunderisotonicconditions.Moreover,ClC-3appearsto beinhibitedbyPKC,whereasPKCstimulatesVSORCl- channelsinmanypreparations.
BCl(ormaxiCl-)channelsthatareactivatedunderhypotonicconditionshavebeenreportedfromcardiacmyocytesofnew-bornrats,ratcorticalastrocytesinprimary culture,therabbitrenalcorticalcollectingductcelllineRCCT-28Aandprimaryculturesofbovinepigmentedciliaryepithelium.Typically,BClchannelsexhibitunitaryconductancesinthe rangeof100pSto400pSandalinearcurrent-to-voltagerelationship.Inmostinstances, uptofiveidenticalsubconductancelevelsweredetected.BClchannelshaveabell-shaped voltagedependencewithverylowactivityoutsidetherangeof–40mVto+40mV.BClchannelsmaybeactivatedby membranestretchorbypatchexcisionfromnonswollencells.Basedontheeffectsofcytochalasins,adirectregulatoryinteractionwiththeactincytoskeletonisassumed.BClchannelsmayberelatedtothevoltage-dependentan-ionchannel(VDAC),aporin-likechannelfoundineukaryoticmitochondriaortothe brain-derivedvoltage-dependentanionchannel1(BR1-VDAC),whichisalsoexpressed inlymphocytes,kidney,heart,andskeletalmuscle.
Hypotonicity-inducedSCl(orminiCl-)channelswithunitaryconductancesinthe rangeof2pSto10pShavebeenreportedfromsingle-channelpatch-clampstudiesonthe ventricularmembraneofchoroidplexuandpigmentedandnonpigmentedbovineciliary epithelium.ItisbecauseoftheselowconductancesthatthebiophysicalpropertiesandtheactualcontributionofSClchannelstomacroscopicconduc-tancesweresometimeshardtodefine.Inthehumancoloncell-lineHT29,forinstance,the Cl- channelsactivatedbycellswellingappearedtobetoosmalltobedetectedbyuseof single-channelpatch-clamptechniques.
K+-Cl-symport
ElectroneutralKClreleaseappearstobeoneofthemajorfeaturesduringRVDinmany systems.
K+-Cl-symportwasfirstdefinedinerythrocytesasCl-dependentandouabain-insensi-tiveK+transportand,thereafter,detectedinmanyothercelltypes.FourisoformsofK+-Cl-symporters(KCC1–KCC4)havebeencloned todatewhichexhibitsignificantmolecularsimilaritiestotheNa+-Cl-symporter(NCC) andtotheknownmediatorsofNa+-K+-2Cl-symport(NKCC1andNKCC2;seethesec-tionentitled“Na+-K+-2Cl-symport”).Alltogether,theyformthesuperfamilyofcation-Cl-cotransporters(CCCs)thatappearstooriginatefromanancientshorttransportprotein foundincyanobacteria.KCC3wasmostprominentinheartand kidney,butalsodetectableinskeletalmuscle,placenta,liver,lung,pancreas,endothelial cells,andinthecentralnervoussystem.
Cationchannels
Insomesystems,hypotonicstressleadstotheactivationofnonselectivecationchannels. Atfirstsightthismaybesurprisingbecause,duetothesteeperelectrochemicalNa+than K+gradientacrossthecellmembrane,nonselectivecationchannelsareexpectedtomediateanetuptakeratherthanareleaseofosmolytes.Ithastobeconsidered,however,that someofthesechannelsexhibitaslightlyhigherpermeabilityforK+whencomparedto Na+.Moreover,certainnonselectivecationchannelsarepermeabletodivalentcationsso thattheymayplayaroleinCa2+signaling.Somenonselectivecationchannelsaresensi-tivetomembranestretch.
Asecondgroupofcationchannelsdoesnotdiscriminatesignificantlyamongsmall monovalentcations,butthesechannelsexhibitasizeablepermeabilitytoCa2+(and/or Ba2+).ChannelsofthistypewerefoundinEhrlichascitestumorcells,rathepatocytes(Bear1990),singleproximaltubulecellsoffrogkidney,porcinecerebralcapillaries,ratatrialcells,guinea-piggastricsmoothmuscle cells,aswellasinRanaandXenopusoocytes.
Recently,firstmolecularcorrelatestohypotonicity-induced,nonselective,andCa2+-permeablecationchannelscouldbeidentifiedinhuman,rat,mouse,andchicken.These arethe“osmosensitive,transientreceptorpotentialchannel4”.
Swellingandstretch-activatedchannelsthatarenonselectiveforsmallmonovalentcationsandapparentlyimpermeabletoCa2+werefoundinthebasolateralmembraneoffrog proximaltubule, andinhumanepididymalcells.Unitaryconductanceswereintherange of7pSto25pS.IntoadurinarybladderandintheA6celllinederivedfromXenopus kidney,cellswellingactivatednonselectivecationchannelsthatwereblockedbyextracel-lularCa2+atmillimolarconcentrations.TheactualroleofthesechannelsintheRVDprocessiscompletelyunclearandre-mainstobeelucidated.
OrganicosmolytesinRVD
Afterexposuretohypotonicmedia,cellsbeginalmostimmediatelytoreleaseorganicos-molytesinadditiontoinorganicosmolytes.Thisreleaseisnottheconsequenceofageneralincreaseofthemembranepermeabilitytoorganicsolutes,butshowsamarkedspecificityandthusinvolvesspecificrelease pathways.Thesepathwaysareonlybrieflyactivatedasexemplifiedforsorbitolefflux fromIMCDcellsinFig.5.
Fig. 5 Time course of activation of sorbitol efflux from IMCD cells during exposure to hypotonic solution. IMCD cells isolated at 600 mOsm/l were exposed at time 0 to a medium of 300 mOsm/l. Membrane permeability was calculated based on the difference between intracellular and extracellular concentration of sorbitol.
Someofthemhavequitelowosmoticthresholds.Insomecells,organicosmolytereleaseaccountsforabout50%ofthetotalosmolytesreleasedduringRVD;thereby,itsimportanceinoverallcellvolumeregulationbecomesevident.Italsobecomesnecessaryfor thecellstocoordinatethevariouschannelactivitiessothattheycanoperateinconcertat theproperactivity.Howthiscoordinationisachievedisoneoftheenigmasthatremainto besolved.
Swelling-activatedtaurinerelease
Taurinereleaseinducedbycellswellingisawell-studiedphenomenoninEhrlichAscites tumorcells,articularchondrocytes,astrocytes,endothelialcells,neurons,andrenalmedullarycells.Taurineeffluxis passiveanddirectedonlybytheconcentrationdifference,lackssaturabilityandtrans-stimulation.Inaddition,taurinefluxinavarietyof cellsisinhibitedbyarangeofanionchannelblockers.Thesepropertiessuggestthattaurineeffluxismediated byatransportsystemwhichismoresimilartoachannelthantoatransporte.Thesearchfor suchchannelshasrevealedthefollowingcandidatesthusfar.
VSOR-Cl-channel,VSOAC,orVRAC
Asdiscussedinmoredetailabove(seethesectionentitled“Cl- channels”)thesechannels areoutwardlyrectifyingandhavesignificantpermeabilitiestoanionicaminoacids,includingthose withanionicsidechains(glutamateandaspartate),aswellastheanionicformofzwitterionicaminoacidssuchastaurine.Attemptstofurtherinvestigatethesubstratespecificity withregardtotheorganicosmolytesbymeasuringtheeffectofextracellularaminoacids onRVDingliacells orbyelectrophysiologicalstudiesrevealedthatthechannelhasahigherpermeabilityto-aminoacidscomparedto-amino acids.
Inavarietyofcells,VSOR-Cl-channelandthetaurineeffluxpathwayshaveanalmost identicalsensitivitytoanionchannelblockers.Figure6showsasanexamplethefindings inratIMCDcells.
Fig. 6 Effects of various anion channel blockers on swelling-activated anion conductance in rat IMCD cells. Inhibition of volume-activated anion conductance under symmetrical taurine conditions (300 nM; pH 7.8; 31 nM charged; %inhibition ITau) plotted against inhibition of anion currents in symmetrical140mMCsClconditions(%inhibitionICl-)byvariousanionchannelblockers(unpairedexperiments);n=5 forallexperimentalconditions.Formostdatapoints,SEvaluesaresmallerthanthesymbolsused.NFA niflumicacid,DDFS1,9-dideoxyforskolin,NPPB5-nitro-2-(3-phenylpropylamino) benzoate,DIDS4,41-di-isothiocyanostilbene-2,2disulfonicacid,SITS4-acetamino-40-isothiocyanostilbene-2,2disulfonicacid.
Becausetherateofactivationisalsosimilar,thesedatasuggestthat chlorideandtaurinesharethesamereleasepath.
Interestingly,VSOACactivityisregulatedbythemetabolicstatusofthecell.WhenintracellularATPfalls,therelativetaurineeffluxdecreases.Thishasbeenobservedinrat glialcells,endothelialcellsandratIMCDcells.
Othersystemsinvolvedinvolumeregulation
Cl-/HCO3-antiport
Thesuperfamilyofbicarbonatetransporters(BTs)consistsoftwogroups,namely,theAE familyofanionexchangers(i.e.,Cl-/HCO3-antiporters)andthefamilyofHCO3-transportersthatarecoupledtoNa+.Whereasnodirect contributionofNBCstocellvolumeregulationhasbeenreportedsofar,Cl-/HCO3-anti-portisofconsiderableimportancefor,bothRVIaswellasRVD.Fourisoformshavebeen
Phospholemman
Phospholemman(PLM)isasmallmembraneproteinof15kDawhichwasfirstpurified, cloned,andsequencedfromdogheart.Overexpressionof PLM in HEK293cells significantlyincreased the membranecurrents elicitedbyhypotonicstressaswellasthereleaseofCl-(I-) andtaurineduringRVD;moreover,thisosmolyterelease couldbemarkedlyreducedbyuseofPLM-specificantisenseoligonucleotides.
TakentogethertheseresultssuggestaroleofPLMinbothRVDandRVI.Thiscould meanthatPLMfunctionsasalastlineofdefensesothatacellunderstronganisotonic conditionsopensalargeosmoticshuntjusttosurvive.
Na+, K+-ATPase
Cellvolumehomeostasisdependsonthebalancebetweenosmolyteentryintothecelland removalofosmolytesfromthecellinterior.Forcellsthatpossesssodiumentrypathways, suchassodiumchannels,Na+/H+antiporter,Na+, K+,2Cl-symporter,orothersodiumcotransporters,sodiumenteringthecellsisremovedbytheprimaryactiveNa+, K+-ATPase. Theactivityofthisenzyme,therebyplaysanimportantroleinmaintainingthecellvolumeasindicatedbythecellswellingobservedinisotonicmediawhenouabainispresentintheincubationmedium.ThisobservationreflectstheactualstoichiometryofNa+, K+-ATPaseof3Na+/2K+whichcontinuouslyleadstoanetlossofcellularinorganicosmolytes.Inthiscontext,itisalsoofnotethatinsomesystemsaninhibitionoftheenzyme appearstodirectlycontributetotheRVIprocesses.
DuringRVIfollowingtheexposureofcellstoshrinkageinhypertonicmedia,theNa+, K+-ATPaseisparticularlyimportantincellsthatprimarily andpredominantlyactivateregulatoryosmolytetransportsystems,whichenhancetheintracellularsodiumcontent,such astheNa+/H+antiporter,theNa+, K+,2Cl-symporter,orasodiumchannel,asdiscussed above.InastudyinwhichosmolyteandNa+transportbalancesofrathepatocyteswere quantifiedas afunction ofhypertonic stress(see Fig.7),itcould be demonstratedthat ouabain-sensitive86Rbuptake,representingthetransportactivityoftheNa+, K+-ATPase,increasedalmostfourfoldinasaturablefashionwithincreasingextracellularosmolarity.
Fig. 7BalancebetweenNa+importandNa+exportbytheNa+, K+-ATPaseplustheactualincreaseofcell Na+.Rathepatocytesinprimaryculturewereexposedtoincreasingosmolarities.Notethesaturabilityof theNa+, K+-ATPasepump rate.
Signaltransductioninvolumeregulation
Sensorysystemsandsetpoints
Althoughthesearchforsensorymechanismsofcellvolumechangeshasbeencarriedout veryintensivelyduringrecentyears,aconclusivemodelofanosmosensorinmammalian cellshasnotyetbeenpresented.Themaincauseforthisgapliesinthenatureofcellular signallingevents.Usually,severalsignallingmechanismsareactivatedinparalleland someofthemdoinfluenceeachothermutually.Thisintegrativecharacterofsignalling networksmakestheassignmentofsignallingeventstodistinctpathwaysdifficult.The conceptthatnotonesingleosmosensorbutratheracomplexsensorysystemexists,hasto betakenintoaccount.Suchasensorysystemhastofulfilltwomajorrequirements.Onthe onehand,ithastobeverysensitivetoperceiveevenslightalterationsofcellvolume.On theotherhand,thesensormustamplifyandtransfertheinformationtotriggersignalling cascadesleadingtotheactivationofvolumeregulatoryprocesses.
Themechanismsthathavebeenreportedtobeimplicatedinsensingchangesofcell hydrationcanbeclassifiedintothreemajorgroups,accordingtotheircellularlocation. Theycanbe(1)associatedwiththecytoskeletonperceivingchangesintheinteractionbe-tweenextracellularmatrixandplasmamembranereceptors;(2)intracellularsolutesensors sensitivetochangesinmacromolecularcrowdingand/orionicstrengthand(3)membrane-basedsensorswhichrecordalterationsofcellmembranestretch.
Extracellularmatrixandcytoskeleton
Cellsadheretotheambientextracellularmatrix(ECM)viacellularintegrinswhichare clusteredtofocaladhesionsuponbindingofECM-ligands.Proteinsthatareassociated withtheseadhesionsitesmayservetomaintainstructuralintegrity,andequallywell,theymaybeinvolvedinsignaltransduction.
Cellmembranestretch
Cellswellingcausesstretchingoftheplasmamembrane,whereasshrinkageleadstoadecreaseincellmembranestretch.Thesealterationsofmembranetension maygenerateasignalperceivedbystretch-sensitivechannels.Amongthestretch-sensitive channels,bothstretch-activatedandstretch-inactivatedchannelscanbefound.Stretch-activatedchannelscaneitherbenonselectivecationchannelspermeableforK+, Na+and Ca2+,orchannelsselectiveforCa2+, K+orCl-(HoffmannandDunham1995;Langetal.
1998a).
Theactivationofnonselectivestretch-activatedcationchannelsuponcellswellingwas demonstratedinseveralcells.Activationofthesechannelscon-tributestoRVD,probablymainlybytheresultingCa2+current(HoffmannandDunham.Theresulting(local)increaseinintracellularCa2+thenactivates Ca2+-sensitiveK+channels(Christensen1987;Ubletal.1988).ThelossofK+isaccompaniedbyosmoticwatermovement,leadingtovolumedecrease(seethesectionsentitled“K+channels”and“Cationchannels”).
Theactivationofselectivestretch-activatedionchannelscontributesdirectlytocell volumeregulation.Stretch-andvolume-sensitiveK+channelsweredemonstratedforNec-turusproximaltubulecells,gallbladdercellsandenterocytes.Similarly,cellmembranestretchmay alsoleadtotheactivationofstretch-sensitivemaxiCl- channels,whichwerereportedto beactivateduponcellswellinginseveralsystems(seethesectionentitled“Cl-chan-nels”).
Stretch-inhibitedchannelsarenotascommonasstretch-activatedchannels,andtheir relevanceforcellvolumeregulationisusuallyregardedasnegligible.Nevertheless,Suzukietal.showed,thatacationchannelcloned fromratkidneyisinactivatedbymembranestretchduetocellswellingandactivatedby cellshrinkage(Suzukietal.1999).
Themechanismbywhichstretch-sensitivechannelsareregulatedbycellvolume changeshasnotyetbeenfullyelucidated.Signalsmodulatingstretch-sensitivechannelcouldbemechanicaltension,conveyedbythemembraneorthecytoskeleton,orthereleaseofstretch-sensitivemessengers,suchasfattyacids.
Lipidderivedmessengers
Phosphoinositides
Lipidsignallingthroughphosphoinositideswasreportedtobeinvolvedincellsurvival, cytoskeletalremodeling,metaboliccontrol,andvesiculartrafficking.Sincealltheseprocessesarerelatedtocellvolumeregulation,itseemsnotsurprisingthatthereisampleevidenceforthein-volvementoflipid-derivedmessengersinprocessesmaintainingosmolytebalance.Phosphoinositidescaninterfereatdifferentlevelswithsignallingcascades.Assignallinginter-mediates,theycanserveassubstrateandcofactorforphospholipasesorphosphatidylino-sitolkinases.TheyarealsocapableofregulatingvariousenzymeslikeRhoGTPasesand profilinandmodulatingionchannelsandtransporters.
Arachidonicacidandeicosanoids
Arachidonicacid(AA)canbedirectlyreleasedfromphosphatidylcholineandphosphati-dylethanolaminebytheactivityofphospholipaseA2(PLA2).Thecleavageofphosphati-dylinositolbyphospholipaseCyieldsarachidonicacidoutofdiacylglycerol(DAG).PhospholipaseDproducesphosphaticacid,whichcanbetransferredtoarachidonicacidbythe actionofdiglyceridelipase.PhosphaticacidmayalsobehydrolysedbyPLA2intoAAand lysophosphaticacid(LPA).
Anincreaseintheintracellularconcentrationofarachidonicacidanditsderivatesupon hypotoniccellswellinghasbeenshownforratIMCDcells(Tineletal.1997),humanneu-roblastomacells(Basavappaetal.1998),andEhrlichAscitestumorcells.ThisreleaseofAAseemstobemainlycausedbytheactivationofPLA2.
Inagreementwiththesefindings,inhibitionofPLA2preventstheactivationofhypotonicallyactivatedeventslikeCl-currentinbovinepigmentedciliaryepithelialcellsandiodideeffluxinratbrainendothelialcells.
Phosphaticacidandlysophosphaticacid
PhospholipaseD(PLD)generatesthelipidsecondmessengersphosphaticacid(PA)and cholinebythecleavageofphosphatidylcholine.OnlyfewexamplesexistfortheinvolvementofPLDinvolume-regulatorysignalling.Inskateerythrocytes,hypotonictreatment increasestheturnoverofphosphatidylcholineandtheformationofPAduetotheactiva-tionofPLD.
Diacylglycerol
Inerythrocytes,theintracellularlevelofdiacylglycerol(DAG)isincreasedbyhypotonicity.DAGisaproductofPLCandabletoactivateproteinkinaseC(PKC).PKCisaserine/threoninekinaseofcentralimportanceinintracellularsig-nallingcascades;hence,itisnotastonishingthatPKCisactivateduponhypotoniccell swellingaswellasuponhypertoniccellshrinkageinvarioussystems.
Cyclicnucleotides
TheintracellularamountsofcAMP,aswellascAMP-dependentphosphorylation,have beendemonstratedtobeunaffectedbyosmoticcellshrinkageinduckandturkeyerythrocytes.InitiationofthecAMPpathwayismore likelytoinhibitRVI,sincecAMP-dependentactivationofPKAcanpreventtheuptakeof theorganicosmolytesbetaineandmyo-inositol,asshownforMDCKcells.
Intracellularcalcium
Aninvolvementofcalciuminvolumeregulationhasbeenfoundinmanycellularsystems. Inparticular,theregulationofcellswellingseemstooftenbeaccompaniedbycalcium signals.Inthecaseofhypertonicstressandcellshrinkage,otherintracellularsignaltransductionprocessesseemtoberesponsiblefortherecoveryofthecellvolumeandthusthe reportsaboutaninvolvementofcalciuminRVIareveryrare.Inthisoverview,wewillfocus,therefore,ontheroleofcalcium duringRVD.
Thecoreprocessofvolumeregulationaftercellswellingisareleaseofosmolytes,re-sultinginreductionofcellularwatercontent.Manydifferenttransportprocessesinvolved inRVDhavebeenfoundtobecalcium-dependent.Calcium-regulatedpotassiumconductanceviaBKCa,IKCa,andSKCahavebeenfoundinmanycells.Alsochlorideconductancesinducedbyvolumechangeshavebeenob-servedtoberegulatedbycalcium/calmodulin-dependentprocesses(seethesectionenti-tled“Cl- channels”).Inguineapigjejunalvillusepithelialcells,acalcium/calmodulin kinaseII-mediatedphosphorylationisacriticaldeterminantofthevolumeregulation.Insomecells,thecalciumactiononthesametransport systemseemstobetransmittedviadifferenteffectorproteins;forexample,inhumancer-vicalcancercells,myosinlight-chainkinaseandproteinkinaseCregulateavolume-sensi-tivechloridechannel.
Thesourceofthecalciuminvolvedinthevolumeregulationseemstobecelltype-dependent.Inseveralcells,RVDstrictlydependsoncalciumentryacrosstheplasmamembrane.Inthesesystems,thevolumeresponsecanbeinhibitedbyextracellularcalciumre-movalorbyadditionofcalciumchannelblockers.Aswelling-inducedriseinintracellular calciuminrabbitmedullarythickascendinglimbcellswasabsentinlow-calciumsolu-tions(seeFig.9).
Conclusionsandperspectives
Thedatapresentedabovedocumentoncemorethecomplexityofeventsthatultimately resultinthemaintenanceofcellvolumeanditsrestorationafterdisturbancebyanosmotic differencebetweenintra-andextracellularspace.Thiscomplexity,althoughdifficultto decipher,providesdifferentcellswithvariousmechanismsthatareadaptedtoprotect themselvesandatthesametimetofulfilltheiroverallroleintheorganism.Withinoneparticularcell,thepresenceofmultiplesystemsmightopenmultiplepossibilitiesto achieveagradedresponsetoosmoticstimuli.
Toobtainfurtherinsightintothiscomplexityseveralstepsatdifferentlevelsofresolu-tionarenecessary.Atthelevelofthesinglemolecules,propermolecularandpharmacologicalidentification,properbiophysicalcharacterization,andproperanalysisofbiochem-icalalterationshavetobecontinuedandintensified.Atthecellularlevel,thesimultaneous measurementoftheactivitiesofthemostimportantosmolytetransportersandtheconcen-trationofthevariousosmolytesatahighertemporalandspatialresolutionhavetobeper-formed.Thesameholdsforthedeterminationofthelocalizationandactivityofthevari-ouselementsofthesignaltransductionnetwork.
Onlythenwillitbepossibletodescribethedynamicnatureofcellvolumeregulation anditsunderlyingmechanismsinaquantitativewaybasedoncausalrelationships.
1